The present invention relates to methods for determining the structure of polypeptides having particular structural and biological activities and affinities.
The systematic design of pharmaceutical agents has currently reached a point where medicinal pharmacologists can often predict the activity of a particular pharmacologic agent from knowledge of its structure/function activity on a chemical level. This knowledge has been particularly useful in the design of new pharmacologic agents which are structurally related to a parent compound, but which exhibit new pharmacologic properties or activities.
For example, in the area of steroid biochemistry and design, the structure of various steroids has been modified in numerous ways to provide for enhanced or specialized activities. Another example of systematic drug design is in the medicinal chemistry of the synthetic penicillins: synthetic penicillins have now been designed which exhibit a number of activities not possessed by the non-synthetic penicillins. These improvements include a conference of oral activity, wide-spectrum activity, and activity against penicillinase-producing bacteria.
However, relatively little is known concerning the structure/function activities of macromolecular structures like proteins. For example, while it is known that antibodies bind to antigens, the underlying attractive interactions are incompletely understood. Even less is known about the underlying mechanism of the response to an antigenic challenge of producing a protein, in the form of an antibody, which is capable of binding an antigen.
Similarly, the interaction of peptide hormones with their hormone receptors is incompletely understood. It is known that in both the binding affinity of the peptide hormone for its receptor and the intrinsic activity of that bound hormone in "stimulating" the receptor, hormonal activity is expressed. From known structure/function relationships of non-protein hormones, it has been postulated that binding activity and intrinsic stimulating activity involve separate structural considerations. Certain chemical structures appear to provide for binding of the ligand, for example, a hormone, to its receptor. Yet other chemical structures appear to provide for "stimulation" of the receptor once the hormone is bound thereto.
Agents which possess binding activity, but not intrinsic stimulating activity, are known as "blockers" or antagonists in that they block the activity of the true hormone. An example of such a blocking agent is isoproterenol, a well-known catecholamine beta-blocker which was designed based on some knowledge of the structure/function relationships of catecholamines with their receptors. Similarly, agonists which both bind and activate hormonal receptors have been produced. No such structure/function relationships are entirely known for the polypeptide hormones. Thus, there is presently no way to accurately enable the systematic design of polypeptides capable of specifically interacting with a particular protein hormone receptor or with a particular polypeptide hormone.
All organisms having an intact immune system possess the biological capability to produce a class of very specialized proteins known as immunoglobulins. Immunoglobulins are produced by specialized cells of an immunocompetent organism in response to the presence of a molecule which is foreign to that organism. These foreign molecules are generally termed antigens. Antigens are operationally defined as being molecules capable of eliciting the formation of a complementary antibody in a given organism. A specific antibody thus formed is capable of binding to the antigen which stimulated its formation. The biological function of a specific antibody is to bind a foreign antigen and thus lead to its inactivation.
Scientists have succeeded in manipulating the immune system of various organisms to provide a vast array of antibodies which have proven useful in both therapeutic and diagnostic medicine. Recently, through the advent of hybridoma technology, science has developed a capability to produce monoclonal antibodies which will bind with specificity to a chosen molecular structure termed the determinant. The usefulness of such specific antibodies is immense, ranging from recent clinical experimentation which suggest an important future role in combating cancer to an everyday clinical role for antibodies in the detection of numerous disease states through blood examination.
One very interesting but largely theoretical application of antibody technology is in the area of anti-idiotypic antibodies. An anti-idiotypic antibody is a second antibody having binding capability for the idiotype or binding site of a first antibody. Such an anti-idiotypic antibody exhibits features in common with the antigen to which the first antibody binds. For example, if one generates antibodies against insulin and then proceeds to generate anti-idiotypic antibodies directed against the anti-insulin antibodies, a portion (idiotype) of the anti-idiotypic antibodies will exhibit insulin-like properties. This finding lends credence to the theory that the binding site of an antibody is a three-dimensional negative-image of the antigen and that an anti-idiotypic antibody to a first antibody is therefore a positive image of the original antigen. Such observations suggest that if such interactive structures could be designed and produced, a whole new array of biologically active substances, for instance, polypeptide hormones or receptors therefor, could be developed which exhibit a wide array of new and useful activities.
Although antibody technology has advanced rapidly, it still has fundamental technological limits. Science and medicine, for example, must still rely on an antibody-producing cell to generate the antibodies. Therefore, scientists have no direct control over antibody production. Such direct control would be a very important advantage. It would allow such advances as the production of man-made "antibodies" that could specifically interact with, or bind, not merely a selected molecule but a preselected portion of that molecule. The underlying basis of the attractive interaction between the antibody and antigen is as yet incompletely understood.
From the foregoing discussion, it is evident that antibody-producing cells have a mechanism to ascertain the chemical structure of an antigen and produce a complementary chemical structure in the form of an antibody. Such complementary results in a capability of binding to the antigenic structure. Prior to the advent of the present invention, in order to design or construct a protein structure complementary to, and thus capable of binding with another protein structure, a knowledge of the chemical interactions which underlie the binding phenomenon was necessary.
All proteins or peptides primarily are polymers of monomeric amino acid units. There are, in general, twenty different amino acids, each possessing a different chemical structure and thus different chemical and physical properties. For example some amino acids tend to be more hydrophobic in nature while others tend to be more hydrophilic in nature. Similarly, some amino acids tend to attract certain other amino acids while repelling yet other amino acids. Therefore, within any given protein, there are a variety of both attractive forces and repulsive forces exhibited by the individual amino acids of that protein. In addition to these interactive forces between amino acids of a given protein, there are also interactive forces between the amino acids and the surrounding environment. The latter forces depend on whether the protein resides, for example, in an aqueous or hydrophilic environment or in a non-aqueous or hydrophobic environment.
The interactive forces exhibited by the amino acids of a given protein are a major factor in determining the three-dimensional, or "ternary", structure of that protein. Therefore, in one view, certain regions within the protein are binding or attracting certain other regions of the same protein while other regions may be repelling certain regions within the protein. The net result is to give each protein a characteristic shape and, therefore, its functional activity.
Recently, there has been developed a means for characterizing amino acids in terms of hydropathy which reflects relative hydrophilicity and hydrophobicity (Kyte et al, (1982) J. Molec. Biol. Vol. 157, pp 105-132). A hydropathy scale was therein derived wherein the hydrophilic and hydrophobic properties of each of the twenty amino acid side-chains was taken into consideration. A computer program was utilized to continuously determine the average hydropathy within a polypeptide sequence of predetermined length. This study demonstrated that proteins have very distinct regions of hydrophobicity and hydrophilicity and that the intramolecular, in addition, of course, to internal disulfide bonding interaction of such regions, can account for the three dimensional structure of the proteins.
An even more recent study has suggested that amphiphilic protein structures, that is, protein structures which contain both hydrophilic and hydrophobic amino acids and regions, play an important role in maintaining the activity of both protein hormones and their receptors (Kaiser et al (1984) Science Vol. 223 pp 249-255). This study further suggests that amphiphilic structures in hormone receptors, for example, might be complementary as a mirror-image of amphiphilic structures in the hormones themselves. Therefore, the interaction between a hormone and its receptor could be mediated by a specific interaction between the amphiphilic structure of the hormone and a complementary amphiphilic structure of the receptor. One way in which this concept may be envisioned is to consider the model concept of a lock and its key, with the lock configuration representing the amphiphilic structure of the receptor and the configuration of the key representing the complementary amphiphilic structure of the hormone agent.
Accordingly, a means of systematically designing polypeptides which are capable of binding or interacting with known peptides, proteins or proteinaceous receptors would be of great utility. For example, practical knowledge concerning the design of receptor-interactive structures of proteinaceous hormones should lead to the development of whole new classes of synthetic hormones with greater specificity of activity. Conversely, one could design and produce polypeptides which are complementary to known proteinaceous hormones and therefore capable of binding to these hormones. Such designed polypeptides may be utilized, for example, to render the complementary hormone inactive.
Similarly, such knowledge of protein or peptide design could prove very significant for many fields of scientific research. For example, if a synthetic polypeptide which is complementary to a protein hormone is structurally analogous to the biological receptor for that hormone, then an antibody directed against that complementary protein should also bind the true hormone receptor. Such antibodies would be useful in studying and isolating specific hormone receptors or portions thereof to thereby lead to an even greater understanding of hormone-receptor interactions. In addition, a synthetic protein or peptide which is complementary to a particular protein should be useful in the crystallization of that protein for the purpose, for example, of probing the protein structure through x-ray crystallography. Further, detoxifying polypeptides could be designed to tightly and specifically bind to toxic peptides found in nature and sometimes ingested.
The above illustrations are just a few of the numerous possible applications that synthetic protein or peptide design capabilities would enable. The ability to systematically design a polypeptide that will interact with or bind to known proteins, the design being based on structural considerations of the known protein, would clearly constitute a scientific breakthrough of major proportions in the field of peptide and/or chemistry and medicinal pharmacology.
For purposes of clarification and consistency, the following terms are defined as to their general meaning herein.
The term antiparallel, referring to nucleic acid pairings, indicates a directionality as to the paired nucleic acids. The original nucleic acid may be in a 5' to 3' direction where the 5' and 3' refer to positions on the sugar moeities involved in nucleotide coupling. The second nucleic acid strand base-paired or complementary to the original nucleic acid strand lies in a 3' to 5' direction when linearly aligned with the original strand having a 5' to 3' directionality.
The coding nucleic acid contains the sequence of nucleotide triplets (codons) specifying a sequence of amino acids when read in a 5' to 3' direction. The noncoding (complementary) nucleic acid (or nucleic acid strand) is complementary to the coding nucleic acid (or nucleic acid strand), the strands lying or base-pairing in an antiparallel direction.
The term hydropathic complementarity, referring to the hydropathic scores (a relative measure of hydrophilicity and hydrophobicity) of amino acids indicates a low hydropathy corresponding to a high hydropathy and vice versa.
In referring to structures comprising amino acids, they are generally referred to as peptides, polypeptides or proteins, this order designating an increase in size between, for example, dipeptides, oligopeptides, and proteins containing many hundred of amino acids.
The term complementary, or complement, as used herein has a meaning based upon its context of usage. For example, complementary bases or nucleotides are those characteristically forming hydrogen bonds (G-C and A-T or A-U), complementary codons nucleic acids or strands thereof are hydrogen bonded polynucleotide components of a double nucleic acid strand such of that in the classically defined double helix for example complementary amino acids usually having hydropathic complementarity are those directed by members of a pair of complementary codons.
Complementary peptides or polypeptides and their related original peptide or protein are a pair of peptides directed by complementary nucleotide or amino acid sequences, and characteristically have a binding affinity between members of a pair. Polypeptides complementary to a peptide or at least a portion of a protein, for example, have a binding affinity for the peptide or protein portion. While peptide binding affinities are incompletely understood, they may, in part at least, be explained by the concept of amphiphilic secondary structure described by Kaiser et al (Science (1984) Vol. 223 pp. 249-255).